Ionizing radiation induces cells with past caspase activity that contribute to the adult organ in Drosophila and show reduced Loss of Heterozygosity

There is increasing recognition that cells may activate apoptotic caspases but not die, instead displaying various physiologically relevant consequences. Mechanisms that underlie the life-or-death decision in a cell that has activated apoptotic caspases, however, are incompletely understood. By optimizing a published reporter for past caspase activity, we were able to visualize cells that survived caspase activation specifically after exposure to ionizing radiation in Drosophila larval wing discs. We found that cells with X-ray-induced past active caspases (XPAC) did not arise at random but were born at specific locations within the developing wing imaginal discs of Drosophila larvae. Inhibiting key components of the apoptotic pathway decreased XPAC number, suggesting that apoptotic signaling is needed to induce XPAC cells. Yet, XPAC cells appeared in stereotypical patterns that did not follow the pattern of IR-induced apoptosis, suggesting additional controls at play. Functional testing identified the contribution of wingless (Drosophila Wnt1) and Ras signaling to the prevalence of XPAC cells. Furthermore, by following irradiated larvae into adulthood, we found that XPAC cells contribute to the adult wing. To address the relationship between XPAC and genome stability, we combined a reporter for past caspase activity with mwh, an adult marker for Loss of Heterozygosity (LOH). We found a lower incidence of LOH among XPAC compared to cells that did not activate the reporter for past caspase activity. In addition, at time points when wing disc cells are finishing DNA repair, XPAC cells show an anti-correlation with cells with unrepaired IR-induced double-stranded breaks. Our data suggest that non-lethal caspase activity safeguards the genome by facilitating DNA repair and reducing LOH after transient exposure to X-rays. These results identify a physiological role for non-lethal caspase activity during recovery from radiation damage.


Introduction
die, instead displaying various physiologically relevant consequence such as axon pruning and reduction 38 of the cytoplasm during sperm maturation (for reviews, (1)(2)(3)(4)). In one variation, a cell may return to life 39 from the brink of apoptotic death in a process called anastasis (2, 5-7), which is seen in cultured cells 40 exposed to a variety of death stimuli including ethanol (8), a broad-spectrum kinase inhibitor

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Staurosporine and a small molecule BCL-2 antagonist (9), or ion beam radiation (10). Removal of death-42 inducing stimuli before apoptosis is completed allows some cells to recover even after displaying such 43 apoptotic features as cleaved caspases, Mitochondrial Outer Membrane Permeabilization, or 44 phosphatidylserine flipping. Cells that underwent anastasis are not only alive but are capable of 45 proliferation and initiating tumors. How cells activate apoptotic caspases but not die is still poorly 46 understood. Dissecting this phenomenon requires the ability to detect cells that activated caspases but 47 did not die. Two genetic reporters for past caspase activity have been described in Drosophila 48 melanogaster (11, 12). CasExpress and Caspase Tracker relies on the transcriptional activator GAL4 with 49 a cell-membrane tether that includes a recognition sequence, DQVD, for effector caspase Drice 50 (Drosophila caspase 3/7 in conjunction with the G-trace lineage tracing system (13)). Upon Drice 51 activation, the tether is cleaved and GAL4 enters the nucleus to initiate the expression UAS-FLP 52 recombinase, which catalyzes a recombination event to cause permanent GFP expression. Both sensors 53 show widespread non-lethal caspase activity in nearly every tissue during Drosophila development (11, 54 12). CasExpress reporter was used in a screen for regulators of developmental non-lethal caspase 55 activity that identified apical caspase Dronc, caspase inhibitor p35, pro-survival signaling component 56 Akt, transcription factor dCIZ1 and Ras (14). One of these, dCIZ1, was shown to promote survival after 57 caspase activation by heat shock, oncogenic stress and X-rays.

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We wanted study cells with past caspase activity induced not by developmental events but by ionizing 60 radiation (IR). IR is used to treat more than half of cancer patients where therapeutic success relies on 61 IR's ability to induce apoptosis. Therapy failure can result if cells initiate but do not complete apoptosis 62 after IR exposure, making it important to understand molecular mechanisms that regulate the 63 production of such cells. The use of CasExpress and Caspase Tracker to detect IR-induced non-lethal 64 caspase activation have been challenging because of strong developmental signals from these activity 84 In our published work with Caspase Tracker, we raised the larvae at 18°C and shifted them to 29°C to 85 inactivate GAL80 ts from 0 to 6h after exposure to 4000R (40Gy) of X-rays (15) (Fig. 1A, T=29°C

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Even with limited (6h) activation of GAL4, unirradiated wing discs from unirradiated larvae show GFP+

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75% of CasExpress>GFP cells in irradiated discs are X-ray-induced while the remainder are from 101 developmental caspase activation. To eliminate the GFP signal from the latter, we tried using lower 102 temperatures to inactivate GAL80 in the protocol in Fig. 1A (Figure 1 E-G'), using CasExpress that shows 103 smaller variance than Caspase Tracker -IR (Fig. 1B). We found that we could go as high as 26°C and still 104 keep the CasExpress>GFP area to near zero in unirradiated discs (Fig. 1G', quantified in B) while IR 105 increased the GFP+ area significantly (Fig. 1H', quantified in B). A control CasExpress sensor with a 106 DQVD->DQVA mutation showed no GFP+ cells even with IR (compare Fig. 1I to H'), indicating that GFP+ 107 cells report effector caspase activity specifically. Returning the larvae back to 18°C immediately after 108 irradiation, that is, without any temperature shift, eliminated GFP+ cells (Fig. 1J), indicating that 109 production of GFP+ cells is GAL4-dependent. Because of low GFP+ area without IR at 26°C, the 'signal to 110 noise' ratio increased to 20-fold, meaning most (>90%) of GFP+ cells +IR discs were cells with X-ray-111 induced past active caspases (XPAC). Subsequent experiments use the protocol in Fig. 1A with T=26°C. discs of different sizes and degree of epithelial folding ( Figure 3A-D, arrowheads). We noticed three 130 trends in the prevalence and the location of GFP+ XPAC cells at 24h after IR. First, smaller discs showed 131 more GFP+ area relative to the disc size than larger, more developed discs (compare Fig. 3D' to A'). A 132 plot of % disc area, that is GFP+ against disc size illustrates this trend ( Fig. 3F '6-7d at IR'). The area 133 serves as surrogate for cell number since wing discs are composed of a single layer epithelium. We 134 quantified clone area instead of clone number to avoid errors in assigning cells to clones, especially as 135 XPAC cells appear close together (Fig 2A-C). By using an early (24h) time point, we are catching XPAC 136 cells soon after birth and before cell division makes a significant contribution to clone area (see 137 preceding paragraph). We interpret the dependence of XPAC prevalence on disc size to mean that as the 138 disc increased in developmental age, X-rays were able to induce fewer cells that activated caspases but 139 survived. This correlation was confirmed by aging the larvae for an additional 24h at 18°C before 140 irradiation. This produced larger, more developed discs. Importantly, the same relationship between 141 disc size and % GFP+ area was seen in the older cohort (Fig. 3F, '7-8d at IR'), except that overall values 142 shifted to the right as expected for older discs. Separating discs based on the absence or presence of 143 epithelial folds, a criterion used before to separate discs according to development (20), led to the same 144 conclusion; more developed discs had fewer XPAC cells (Fig. 3G).

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Second, the distribution of GFP+ cells in each disc was not homogenous. The notum (future body wall or  158 what keeps cells alive after they began to activate apoptotic caspases. We used also heterozygotes for 159 the H99 chromosomal deficiency that removes hid, rpr and grim and has been shown to reduce and 160 delay IR-induced apoptosis (16); the effect of H99 would be throughout the organism and not just cell-161 autonomous or conditional. We analyzed discs with no folds because their robust XPAC incidence 162 (~30%) provides room to detect changes up or down. Both, increasing and decreasing apoptotic 163 signaling reduced XPAC prevalence ( Fig 3H). The seemingly paradoxical result can be explained by the where we see no XPAC cells (Fig 4C-C''). Specifically, we noted robust mitotic activity in the notum 185 (arrows) and the pouch (between dashed lines) that includes the future ZNC. Second, the notum and the 186 pouch have similar cell doubling times (20), yet, as the wing disc develops, XPAC cells are lost from the 187 notum before they are lost from the pouch (Fig 4B''). We conclude that cell cycle exit is unlikely to be 188 the cause for XPAC reduction that accompanies development.

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The possibility is that X-rays were less efficient at inducing caspase activity in more developed discs is 191 also unlikely. Cleaved active Dcp1 is absent without irradiation (S1 Figure) but is readily discernable in indicate the hinge cells that we showed before are refractory to IR-induced apoptosis (24)). We 197 conclude that while apoptotic caspase activity is necessary to generate XPAC cells (Fig 3), it is not

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(18, 24). To assay for long term effects of non-lethal caspase activity under these conditions, we 228 followed XPAC cells into adult wings where they appear in an IR-dependent manner (Fig. 6) regardless of 229 larval age at the time of irradiation (6-7d or 7-8d; corresponding to larval XPAC data in Fig. 3F). This 230 allows us to use the multiple wing hair adult marker to quantify Loss of Heterozygosity (LOH), a type of 231 genome aberration. LOH of recessive mwh alleles results in homozygous mutant cells with more than 232 one hair each due to defective actin organization (27). We showed previously that mwh LOH is absent 233 without IR but is induced when larvae are irradiated with 4000R of X-rays (28). At appropriate 234 magnification, it is possible to assign GFP and mwh status to each cell (Fig. 6H). We found that mwh 235 incidence was lower for GFP+ relative to GFP-areas of the same wing (Fig. 6I).

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There are two possible interpretations of the data that cells with XPAC show reduced LOH. Non-lethal 238 caspase activity may facilitate DNA repair to reduce genome instability or non-lethal caspase activity and 239 chromosome breaks may act in a synthetic lethal combination to take the cell above the threshold for

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CasEx>GFP, which becomes detectable at similar times, show mutual exclusion with g-H2Ax; cells that 247 show both are rare (arrowheads in Fig 6J''''). We interpret these results to mean that XPAC cells 248 complete DNA repair more efficiently than their GFP-counterparts. More efficient repair in XPAC cells,

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we propose, could explain the observed reduction in LOH in the adult wing.

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We report here a study of cells with X-ray induced past active caspases (XPAC) during wing development 253 in Drosophila. An optimized temperature shift protocol allowed us to minimize cells with spontaneous, 254 developmental caspase activity and to focus specifically on XPAC cells. Such cells, we found, were able to 255 proliferate and contribute to the adult organ and show reduced genome aberrations as measured by 256 LOH, which results primarily from aneuploidy of whole or segments of chromosomes in Drosophila (29).

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In mouse embryo fibroblasts and immortalized human epithelial cells and fibroblasts (MCF10A and 258 IMR90) exposed to 56 F ion particle radiation, caspase activity, γ-H2Ax signal and DNA fragmentation  ., (18, 24)). We interpret our data to 266 mean that non-lethal caspase activity protects the genome from X-ray-induced chromosome breaks.

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Unlike with 56 F radiation, X-ray-induced DSBs are cleared within 24h, even with much higher IR doses of the genome to allow access to oncogenic transcription factors such as KRAS/E1A used in these studies transformation factors in the wing disc. In other words, consequences of non-lethal caspase activity may 274 depend on the cellular context, specifically, which proteins are present and active to access the DNA. In 275 cells predisposed to transformation such as immortalized cell lines, the outcome may be transformation.

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In cells that lack transformation factor but are robustly expressing DNA repair enzymes, improved repair 277 may be the outcome.

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A prior study used the CasEx>GFP reporter and found that Dronc and p35 affect developmental non-  contrast, Ras V12 decreased the XPAC area by 2-fold (Fig 5F), which we attribute to its ability to prevent 285 caspase amplification like Wg, Dronc DN and p35 (Fig. 3H). We conclude that retention of cells with past 286 caspase activity shows common as well as different regulatory controls depending on whether cells are 287 subject to developmental or IR-induced caspase activity.

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Drosophila stocks are listed in S1 Table and

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After crossing virgins and males, the adults were fed on Nutri-Fly German Formula food (Genesee 303 Scientific) for 3 days at 25°C to boost egg production, before transferring them to 18°. Embryos were 304 collected and larvae were raised on Nutri-Fly Bloomington Formula food (Genesee Scientific). The 305 cultures were monitored daily for 'dimples' on the food surface that denotes crowding and split as 306 needed. Larvae in food were placed in petri dishes and irradiated at room temperature (rt) in a Faxitron food were placed in new vials in temperature bath filled with Lab Armor beads (ThermoFisher), which 309 we found gave more consistent and reproducible data than water baths. After the temperature shift to 310 inactivate GAL80, larvae were returned to 18°C until dissection.

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Wing discs and wings were imaged on a Leica DMR compound microscope using a Q-Imaging R6 332 CCD camera and Ocular or Micro-Manager software. Images were processed and analyzed using ImageJ      The larvae of the genotype CasExpress/G-trace; GAL80 ts /+ were treated as in Fig. 1A where T=26°C.

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The arrows point to the notum and the * marks the distal pouch.

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(I) Percent of image area that is mwh was quantified from images such as G, for the GFP+ and 525 GFP-portion of the surface in focus. The data include wings from animals irradiated as 6-7d (gray) or 7-526 8d (black) old larvae. Each dataset also shows a significant difference in mwh between GFP-and GFP+ 527 areas: p= 0.04 for 6-7d and 0.03 for 7-8d. p-values were computed using a 2-tailed t-test.

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Larvae of the genotype CasExpress/G-trace; GAL80ts/+ were treated as in Fig. 1A where T=26°C. Wing 538 discs were dissected at 4h after exposure to 0 or 4000R of X-rays, fixed, and stained for DNA and with 541 542 S1 Table. Fly stocks used. S1 Figure. Caspase cleavage without IR and DNA double strand breaks with and without IR. Larvae of the genotype CasExpress/G-trace; GAL80ts/+ were treated as in Fig. 1A where T=26°C. Wing discs were dissected at 4h after exposure to 0 or 4000R of X-rays, fixed, and stained for DNA and with antibodies to cleaved Dcp1 (A-A') or g-H2Av (B-C'). The scale bar = 120 microns in A and 24 microns in B-C. S1